Mapping of the nei transcript by RT–PCR. (A) The arrows numbered 2, 7 and 8 represent the primers used in PCR. The arrow numbered 19* represents the primer used to reverse transcribe E.coli total RNA. Primer nei2 anneals 53 bp 3′ to the nei start codon; nei7 anneals 3059 bp 5′ to the nei start codon; nei8 anneals 3536 bp 5′ to the nei start codon. (B) The primer sets used in PCR are indicated above the lanes. Lanes 1 and 4 are positive controls with E.coli genomic DNA as the PCR template. Lanes 2 and 5 are minus reverse transcriptase. Lanes 3 and 6 show PCR results obtained with cDNA from RT with primer 19* (RT19) as the template. The size of the PCR products are indicated in base pairs to the right.

Mapping of the nei transcript by RPAs. (A) The thick lines designated 1–7 indicate the approximate annealing locations of the probes used in the RPAs. The thin lines indicate the products obtained from RPAs with these probes along with the sizes of the products. The sizes and exact annealing locations of the probes are as follows: probe 1, 518 nt, anneals from 90 bp 3′ to the ybgI start codon to 428 bp 5′ to the ybgI start codon; probe 2, 804 nt, anneals from 146 bp 3′ to the ybgJ start codon to 50 bp 3′ to the ybgI start codon; probe 3, 728 nt, anneals from 87 bp 3′ to the ybgK start codon to 10 bp 3′ to the ybgJ start codon; probe 4, 994 nt, anneals from 81 bp 3′ to the ybgL start codon to 21 bp 3′ to the ybgK start codon; probe 5, 853 nt, anneals from 94 bp 3′ to the nei start codon to 12 bp 3′ to the ybgL start codon; probe 6, 578 nt, anneals from 263 bp 3′ to the nei stop codon to 315 bp 5′ to the nei stop codon; probe 7, 848 nt, anneals from 533 bp 3′ to the nei stop codon to 315 bp 5′ to the nei stop codon. (B–D) The probes used in the RPAs are indicated and lane M contains RNA size markers. Lanes 1, 3 and 5 in (B) and lanes 1 and 3 in (C) and (D) show the full-length probe (probe hybridized with yeast RNA and mock digested; only part of the reaction was loaded so that the signal would not overpower the other lanes). Lanes 2, 4 and 6 in (B) and lanes 2 and 4 in (C) and (D) show RPA results. Numbers beside the panels are in base pairs.

Mapping of nei transcription initiation sites by primer extension analysis. (A) Arrows indicate primers used in primer extension analysis. nei16 anneals 95 bp 3′ to the predicted ybgI start codon; nei17 anneals 93 bp 5′ to the predicted ybgI start codon. (B) Lane 1, primer extension with primer nei17; lane 2, primer extension with primer nei16. The size of the products are indicated in base pairs to the left of the figure.

Mapping of the nth transcript by RT–PCR. (A) The arrows numbered 20, 19 and 22 represent the primers used in PCR. The arrows numbered 2* and 5* represent the primers used to reverse transcribe E.coli total RNA. Primer nth20 anneals 88 bp 3′ to the predicted ydgL start codon, nei19 anneals 236 bp 3′ to the predicted ydgK start codon and nei22 anneals 242 bp 5′ to the predicted ydgT start codon. (B) Lanes 1, 4, 10 and 13 are positive controls with E.coli genomic DNA as the PCR template. Lanes 2 and 5 are negative controls with RT reaction mixtures minus reverse transcriptase with primer 2* as the PCR template. Lanes 11 and 14 are the negative controls for primer 5*. Lanes 3 and 6 show PCR results with the cDNA from reverse transcription with primer 2* (RT2) as template. Lanes 12 and 15 show PCR results obtained with the cDNA from RT with primer 5* (RT5) as the template.

Mapping of the nth transcript by RPAs. (A) The thick lines designated 1–7 indicate the approximate annealing locations of the probes used in the RPAs. The thin lines indicate the products obtained from RPAs with these probes along with the sizes of the products. The sizes and exact annealing locations of the probes are as follows: probe 1, 835 nt, anneals from 316 bp 3′ to the ydgK start codon to 519 bp 5′ to the ydgK start codon; probe 2, 504 nt, anneals from 199 bp 3′ to the ydgL start codon to 305 bp 5′ to the ydgK start codon; probe 3, 1259 nt, anneals from 199 bp 3′ to the ydgL start codon to 519 bp 5′ to the ydgK start codon; probe 4, 685 nt, anneals from 113 bp 3′ to the ydgM start codon to 10 bp 3′ to the ydgL start codon; probe 5, 719 nt, anneals from 150 bp 3′ to the ydgN start codon to 3 bp 3′ to the ydgM start codon; probe 6, 866 nt, anneals from 907 bp 3′ to the ydgN start codon to 42 bp 3′ to the ydgN start codon; probe 7, 826 nt, anneals from 1687 bp 3′ to the ydgN start codon to 862 bp 3′ to the ydgN start codon. (B–E) Lanes 1 and 3 in (B), (D) and (E) and lane 1 in (C) show the full-length probe. Lanes 2 and 4 in (B), (D) and (E) and lane 2 in (C) show RPA results.

Mapping of the nth transcript by RPAs. (A) The thick lines designated 8–12 indicate the approximate annealing locations of the probes used in the RPAs. The thin lines indicate the products obtained from RPAs with these probes along with the sizes of the products. The sizes and exact annealing locations of the probes are as follows: probe 8, 805 nt, anneals from 207 bp 3′ to the ydgO start codon to 598 bp 5′ to the ydgN start codon; probe 9, 814 nt, anneals from 118 bp 5′ to the ydgP start codon to 132 bp 3′ to the ydgO start codon; probe 10, 908 nt, anneals from 80 bp 3′ to the ydgQ start codon to 204 bp 5′ to the ydgP start codon; probe 11, 792 nt, anneals from 97 bp 3′ to the nth start codon to 4 bp 3′ to the ydgQ start codon; probe 12, 700 nt, anneals from 372 bp 3′ to the nth stop codon to 328 bp 5′ to the nth stop codon. (B–D) Lanes 1 and 3 in (B) and (C) and lane 1 in (D) show the full-length probe. Lanes 2 and 4 in (B) and (C) and lane 2 in (D) show RPA results.

Mapping of nth transcription initiation sites by primer extension analysis. (A) Arrows indicate primers used in primer extension analysis. nth20 anneals 88 bp 3′ to the predicted ydgL start codon; nth26 anneals 99 bp 3′ to the predicted ydgK start codon. (B) Primer extension with primer nth26. (C) Primer extension with primer nth20

Conservation of gene arrangement for the nei operon. The numbers in the boxes are percent identity/percent similarity of the proteins relative to the E.coli proteins. The numbers between the boxes represent the number of base pairs in the intergenic regions; negative numbers indicate that the ORFs overlap.

Conservation of gene arrangement for the nth operon. Question marks indicate that it was not possible to determine the number of base pairs in the intergenic region.